The objectives of the research to be performed in the next year are to develop shuttle vectors from the endogenous nuclear Dictyostelium discoideum plasmids, to begin application of the shuttle vectors to genetic analysis of D. discoideum and to continue characterizataion of the basic biological features of the endogenous plasmids. Selectable markers including G418 resistance, a dominant cobalt resistance character from D. discoideum, or dominant drug resistance genes from yeast or E. coli will be incorporated in plasmid-based shuttle vectors. If time permits, we will attempt to clone tsg+ alleles that complement a set of previously characterized D. discoideum temperature sensitive conditional-lethal mutants. These mutations are thought to affect the cytoskeleton. Cotransformation will be used to transfer Ddp4 and Ddp5 into strains that can be manipulated by the standard parasexual genetic techniques; this will facilitate the characterization and use of Ddp4 and Ddp5 as cloning vectors. Restriction site maps and cloned probes will be developed for plasmids Ddp3, Ddp4 and Ddp5. Confirmation of the nuclear location of the plasmids by chromatin analyses using micrococcal nuclease will be extended to the remaining plasmids. DNA base compositions of the plasmids will be determined by Tm analysis. Plasmid maintenance and compatibility studies involving haploid, diploid and transformed cells using our rapid screening technique and determination of copy number by hybridization will continue. Characterization of the dominant cobalt resistance phenotype will be emphasized. We will attempt to clone a 7Kb TaqI restriction fragment that is correlated with the presence of this trait and utilize this DNA in analysis of the amplified DNA, its chromosomal location and usefulness as a selectable resistance marker. Expression of plasmid sequences and of the cobalt resistance during the vegetative and developmental stages of the life cycle will be studied uisng Northern blots. We envisage that at the end of the next year of support that publication of manuscripts will be possible on characterization of the D. mucoroides plasmids, on the nuclear location of the Dictyostelium plasmids, on the maintenance and compatibility of the D. discoideum plasmids, and on the dominant cobalt resistance character.